Multiloop engineered heart muscle tissue

ABSTRACT

The invention is directed to a method for the preparation of a multiring engineered heart tissue construct suitable for use in cardiac tissue augmentation and/or replacement therapy. The invention further refers to multiring EHT constructs which comprise at least two force-generating engineered heart tissue rings fused with each other and a device for preparing the same. Finally, the invention relates to force-generating engineered heart tissue rings derived from human cells and their use in drug screening and target validation assays.

FIELD OF THE INVENTION

The invention is directed to a method for the preparation of a multiringengineered heart tissue construct suitable for use in cardiac tissueaugmentation and/or replacement therapy. The invention further refers tomultiring EHT constructs which comprise at least two force-generatingengineered heart tissue rings fused with each other and a device forpreparing the same. Finally, the invention relates to force-generatingengineered heart tissue rings derived from human cells and their use indrug screening and target validation assays.

BACKGROUND OF THE INVENTION

Myocardial infarction and heart failure represent the main cause ofdeath in industrialized countries. The loss of terminally differentiatedcardiac myocytes which is associated with these pathologies accounts fora decrease in myocardial function which can lead to total organ failureor trigger compensatory mechanisms like hypertrophy of the remainingmyocardium, activation of neurohumoral systems, and autokrine/parakrinestimulation by various growth factors/cytokines.

It has been shown that endogenous regenerative mechanisms do not sufficeto compensate for cardiac myocyte death after myocardial infarction.Modern pharmacotherapy can delay, but not reverse the natural course ofthe disease. Thus, exogenous regenerative strategies including cardiacimplantation or coronary transfusion of cells, activation of endogenouscardiac regeneration by pharmacological means, or implantation ofperformed, engineered cardiac tissues have gained increasing attention(Reinlib, L. & Field, L., Circulation 101, E182-7 (2000); Zimmermann, W.H. & Eschenhagen, T., Heart Fail Rev 8, 259-69 (2003); Olson, E. N., NatMed 10, 467-74 (2004); Orlic, D. et al., Proc Nati Acad Sci USA 98,10344-9 (2001)).

So far, cells of various origins and developmental stages have beengrafted into healthy and diseased hearts, including immature cardiacmyocytes (Reinecke, H., Zhang, M., Bartosek, T. & Murry, C. E.;Circulation 100, 193-202 (1999); Muller-Ehmsen, J. et al., Circulation105, 1720-6 (2002); Li, R. K. et al., Circulation 96, 11-179-86;discussion 186-7 (1997)), skeletal myoblasts (Taylor, D. A. et al., NatMed 4, 929-33 (1998); Menasche, P. et al., Lancet 357, 279-80 (2001)),fibroblasts (Etzion, S. et al., Circulation 106, 1125-30 (2002); Sakai,T. et al., J Thorac Cardiovasc Surg 118, 715-24 (1999)), endothelialcells (Condorelli, G. et al., Proc Natl Acad Sci USA 98, 10733-8(2001)), embryonic stem cell-derived cardiac myocytes (Soonpaa, M. H.,Koh, G. Y., Klug, M. G. & Field, L. J., Science 264, 98-101 (1994)), anda number of potential cardiac progenitors from peripheral blood (Assmus,B. et al., Circulation 106, 3009-17 (2002)) or bone marrow (Orlic, D. etal., Nature 410, 701-5 (2001); Stamm, C. et al., Lancet 361, 45-6(2003); Strauer, B. E. et al., Circulation 106, 1913-8 (2002)). Eventhough the evidence for the formation of true cardiac muscle tissue,electrically coupled to the host myocardium is sparse, most studiesreported procedure-induced improvement of contractile function aftercryo-injury or myocardial infarction.

A different concept in cardiac regeneration is to graft heart muscleproduced ex vivo by tissue engineering. Tissue engineering aims atgenerating functional 3-dimensional tissues outside of the body that canby tailored in size, shape and function according to the respectiveneeds before implanting them into the body. In contrast to cellimplantation in preexisting structures, this approach may allow forcomplete replacement of diseased myocardium or reconstitution of missingcardiac structures in individuals with cardiac malformations.

Despite its potential, cardiac tissue engineering is still in itsinfancy for several reasons: (i) Postnatal cardiac myocytes do not ornot sufficiently replicate. Given the high absolute numbers of cellsneeded for cardiac regeneration (Gepstein, L., Circ Res 91, 866-76(2002)), utilisation of primary cardiac myocytes will not be feasible.(ii) To repair myocardial infarctions or correct heart defects, hearttissue patches need to be engineered at a size and with contractilefeatures that have a realistic chance to lend significant support tofailing hearts. Size as well as function and in vivo survival dependcrucially on metabolic supply. Thus, vascularization is likely to be aprerequisite for successful creation and grafting of large cardiactissue patches. (iii) The heart not only needs systolic strength, butalso depends on unimpeded diastolic relaxation. Consequently, engineeredheart muscle must exhibit a large compliance, a feature often neglectedin classical, scaffold-based tissue engineering approaches. (iv)Finally, any successful tissue engineering concept will depend onstructural and electrical integration of implanted tissue into the hostmyocardium. Recent progress in stem cell research has opened newperspectives for cell sourcing, e.g. from embryonic (Kehat, I. et al., JClin Invest 108, 407-14 (2001)) or adult stem cells (Beltrami, A. P. etal., Cell 114, 763-76 (2003); Messina, E. et al., Circ Res (2004)).

Several publications describe the generation of engineered heart tissuein different geometrical sizes and shapes (Zimmermann, W. H. et al.,Circ Res 90, 223-30 (2002); Fink, C. et al., Faseb J 14, 669-79 (2000);see also published International Application WO 01/55297). It was shownthat engineered heart tissue can be reconstituted by mixing heart cellsfrom rat (including cardiac myocytes, fibroblasts, smooth muscle cells,endothelial cells, macrophages and other cells of leukocytic origin,etc.) with collagen type I, matrigel and serum-containing culture medium(the complete mixture is referred to as reconstitution mix).Specifically, ring-shaped casting molds were used to form ring-shapedEHTs. The engineered tissue rings resemble intact heart tissues in termsof force-frequency behaviour, force-length relationship (FrankStarlingmechanism) and response to extracellular calcium (Eschenhagen T, et al.,Faseb J. 11, 683-94 (1997); Zimmermann W H et al., Biotechnol Bioeng.68, 106-14 (2000). These data support the conclusion of true hearttissue-development in vitro from a functional point of view. However,differences exist as well. For example, absolute contractile forces ofEHTs remain lower than in the intact heart. In the literature, maximaltwitch tensions of about 1-2 mN/mm² are reported in artificial heartmuscle (overview in Zimmermann W H, et al., Heart Fail Rev. 8, 259-69(2003)). In comparison, papillary muscles from rat develop 4-10 mN/mm².The difference of EHT force generation as compared to mature heartmuscles most likely reflects both a quantitative and a qualitativeaspect, as for example a lower fractional occupancy of the EHT tissuesby cardiac myocytes and the lower degree of sarcomere development. Infact, single adult cardiac myocytes develop up to 56 mN/mm² These idealforces are unlikely to be reached in intact muscle preparations due tothe lack of oxygen and metabolite supply in the absence of bloodperfusion.

It has been demonstrated that a simple scale-up of the preparationapproach, e.g. by use of larger casting molds than those described inInternational Application WO 01/55297 cannot solve the problem, sincesize of the engineered tissue constructs appears to be limited bymaximum diffusion distances for nutrients and oxygen. Indeed, none ofthe various tissue engineering approaches developed today generatecardiac tissue-like, contracting constructs of a thickness of more than0.8 mm (Zimmermann, W. H. et al., Circ Res 90, 223-30 (2002)). Yet, sofar developed artificial heart tissues do not represent homogeneousmyocardium, but consists of a large fraction of cell-free matrix andinterconnected cardiac muscle strands that do not exceed 20-100 μm inthickness. Interconnected muscle strands can be observed and evaluatedby confo-cal laser scanning microscopy e.g. after actin or actininstaining as outlined in Zimmermann et al. (Circ Res 90, 223-30 (2002)).

Therefore, a need exists for the provision of improved heart tissuegrafts of larger size which overcome the above shortcomings. It has beenan object of the present invention to provide heart tissue grafts havinga size and contractile strength which allows for the effective supportof failing hearts in a mammal, such as a human. The present inventionprovides for a solution of this problem and offers other relatedbenefits as well.

It has surprisingly been found in course of the invention that largethreedimensional tissue grafts which consist of a well-organized andhighly differentiated cardiac muscle syncytium can be produced by fusingtwo or more engineered heart tissue rings with each other. Fusing, asused herein, means that single EHT units or stripes grow together at aregion where sustained physical contact is established to form acontiguous cell assembly. The assembly represents a structurally,electrically and functionally syncytium that may be finally used as acardiac tissue graft in vivo. Such graft constructs are designated“multiring engineered heart tissue construct” herein. Specifically, ithas been found that several engineered heart tissue rings or comparablegeometric forms can be woven together, thus forming a network in whicheach individual construct remains accessible for unlimited diffusion andexchange of nutrients. As a consequence, these constructs do not exhibitthe typical size limitations known from the prior art. Multiringengineered heart tissue constructs of the invention can be produced byusing a plurality of distinct engineered heart tissue rings as thosedescribed in the prior art. The rings can be fused in any suitablemanner to provide “chainmail”-like constructs. By this technique, theform of the multiring constructs can be adjusted dependent on thespecific therapeutic approach. For example, large multiring netlikepatches can be generated for replacing dysfunctional areas of the heartof a mammal. Alternatively, bag-like forms may be generated whichsurround the complete organ. The size of the multiring construct patchescan be varied dependent on the size of the area to be treated. Themultiring engineered heart tissue construct of the invention can becomprised of force-generating engineered heart tissue rings derived fromany mammal. In view of its intended use in the field of tissueaugmentation, it is preferred that the engineered heart tissue rings arederived from human cells.

Thus, according to a first aspect of the invention, an ex vivo methodfor the preparation of a multiring engineered heart tissue constructsuitable for use in cardiac tissue augmentation and/or replacementtherapy is provided, which method comprises the steps of

-   -   a) providing force-generating engineered heart tissue rings;    -   b) placing into contact at least two force-generating engineered        heart tissue rings so that each force-generating engineered        heart tissue ring has one or more contact points to an adjacent        force-generating engineered heart tissue ring; and    -   c) culturing the force-generating engineered heart tissue rings        under conditions which allow fusion of the at least two        force-generating engineered heart tissue rings at the one or        more contact points to form a multiring heart tissue construct.

As used herein, the term “multiring engineered tissue construct” meansan ex vivo produced tissue construct comprised of at least two, andpreferably more than two distinct force-generating engineered hearttissue rings, which may have been produced by tissue engineeringtechniques, such as those described in Zimmermann, et al., Circ Res 90,223-30 (2002) and International Application WO 01/55297. The term“tissue” refers to a group of similar cells united to perform a specificfunction or a grouping of cells that are similarly characterized bytheir structure and function. The constructs of the invention consist ofat least two distinct engineered heart tissue rings. In this context,the expression “multiring” is meant to emphasize that the fusedconstruct comprises a plurality of loop structures originating from theessentially circular shape of the force-generating engineered hearttissue rings that serve as a basis material for the construct of theinvention. Such loop structures have been proven very useful for thehandling of the tissue construct, for example when surgically fixing theconstruct to the recipient organ or tissue. Additionally, the termmultiring is also intended to comprise embodiments in which tissueconstructs are formed which consist of tube- or stripe-like structures.The former might be obtained by establishing side-to-side connectionsbetween two or more rings (FIG. 8B). The latter might be obtained byforming a ring structure and subsequently cut the latter in two or morepieces for further use. An example of such tissue construct is providedin FIG. 8C.

The multiring construct according to the present invention comprisesforce-generating engineered heart tissue rings. The production of suchcontractile engineered heart tissue in circular/annular shape has beendescribed in detail in the prior art, for example in publishedInternational Application WO 01/55297 and in Zimmermann et al., 2000,Biotechnology and Bioengineering Vol. 68, pages 106 to 114. Cardiacmyocytes such as those obtained from tissue samples of neonatal heartsof different mammals may be used for preparing the EHT rings, asdescribed in Zimmermann, et al., Circ Res 90, 223-30 (2002) andInternational application WO 01/55297. As can be taken from thesepublications, engineered heart tissue rings can be obtained by culturingcardiac myocytes in ring-shaped molding devices. It has been shown thatmixed populations consisting of almost identical portions of cardiacmyocytes and non-cardiac myocytes result in EHTs with increasedcontractile properties compared to selected cardiac myocytes (Zimmermannet al. (2003), Heart Failure Rev., 8, 259-269). Fetal or neonatalcardiac myocytes from mammals, such as rat, mouse or primates are asuitable source for obtaining EHT rings. In contrast to differentiatedcardiac myocytes, these cells still have the capability to divide aswell as to redifferentiate after dedifferentiation. Briefly, a solutionof a scaffold substance (such as collagen type I) is mixed with culturemedium (final concentration in the mixture: 1×DMEM; 2% chicken embryoextract; 10% horse serum; 100 jig/ml Streptomycin; 100 U/ml penicillin).The pH of the mixture is adjusted to physiologic values . . . 7.4 with0.1 N NaOH. Engelbreth-Holm-Swarm tumor exudate (also known as“Matrigel”) is added to give a final concentration of 5-15%. Thismixture is added to a cell suspension of fetal or neonatal cells (forexample, 2.5×10⁶ cells/EHT). Alternatively, serum-free media may beused. A serum-free medium suitable for culturing the cardiac myocytes isexemplified in FIG. 12. The person skilled in the art will appreciatethat several modifications as to the components of the medium can beperformed. Moreover, Matrigel may be substituted against insulin andtriiodothyronine, as described in more detail below.

Alternatively, mammalian cardiac myocyte progenitor cells, such aspluripotent stem cells, adult or embryonic, provide a source forpreparing the EHT rings. Preferably, the EHT rings to be used in themethod according to the invention are derived from human cells. Humanengineered heart tissue rings may be obtained by use of circular-shapedcasting molds. In this case, cardiac myocyte progenitor cells, such asstem cells, more preferably pluripotent embryonic stem cells, may beused for preparing the force-generating engineered heart tissue rings.However, also other types of stem cells, such as pluripotent adult stemcells may be used. The preparation of contractile EHT rings derived fromhuman stem cells is particularly described in example 6 of the presentinvention.

The force-generating engineered heart tissue rings produced from humanor non-human mammalian cells comprise cardiac myocytes. However, theyare usually not composed purely of cardiac myocytes but comprise almostall cells species that are normally found in the heart including cardiacmyocytes, fibroblasts, smooth muscle cells, endothelial cells,macrophages and other cells of leukocytotic origin. Typically, thepresence of cardiac cells within a given EHT ring can be confirmed bypositive staining with anti-cardiac myosin heavy chain, anti-α-actinin,anti-desmin and/or anti-cardiac troponin I antibodies (see Kehat I. etal, supra). If cells are derived from human cells, this can readily beconfirmed by well known methods, such as PCR analysis based on typicallyconserved regions of the genome.

The term “ring” as used in the context of the present invention is meantin a broad sense to comprise also geometric forms which do not representa perfect geometric ring. For example, it may also comprise other formswhich consists of annularly closed tissue tube structures. It has beenfound that a circular geometry of the EHT is ideal for several technicaland biological reasons. For example, circular heart tissue structuresallow large scale production with minimal handling. Most importantly,circular engineered heart tissue structures can be easily miniaturizedfor high-throughput screening and they exhibit better tissue formationthan the non-circular designs, since the circular form causeshomogeneous force distribution throughout the tissues. Furthermore,nutrient distribution is facilitated in circular-shaped tissues. Alsonot being particularly restricted to a specific limited size, it hasbeen shown that engineered heart tissue rings having an outer diameterof 8-12 mm have particularly preferred characteristics. The innerdiameter of these rings should be in the range of 6-10 mm. Particularlypreferred are EHT rings with an outer diameter of about 10 and an innerdiameter of about 8 mm (see International Application WO 01/55297).

As used herein, the term “force-generating” means that the engineeredheart tissue rings are able to actively contract against a givenmechanical load. The intrinsic property of heart cells to formcontractile aggregates has been known in the field for years. Severalpublication reported that contractile properties are maintained whenthreedimensional EHTs, such as EHTs of circular shape, are constructed.Contraction of the tissue constructs are triggered by addition of Ca²⁺ions or isoprenaline to the culture medium. Zimmermann, W. H. et al.,Circulation 106, I 151-7 (2002), reported determination of contractilefunctions of engineered tissue rings by isometric force measurements inorgan baths before implantation (see FIG. 2a of the above publication).Maximal twitch tension (TT) values of about 0.4 mN to 0.7 mN weremeasured.

According to a first step of the method for the preparation of themultiring engineered heart tissue construct of the invention, distinctforce-generating engineered heart tissue rings are provided. These ringsare, for example, obtainable by culturing suitable cells in ring-shapedmolding devices. The preparation of such rings from mammalian cellsources is described in the art, for example, in Zimmermann, et al.,Circ Res 90, 223-30 (2002), in International Application WO 01/55297 andin Example 1. Using the same casting molds, engineered heart tissuerings may also be prepared from human stem cells, for example, asdescribed in Example 6.

Subsequently, at least two force-generating engineered heart tissuerings are positioned into contact, so that each of theseforce-generating engineered heart tissue rings has one or more contactpoints with at least one further EHT ring in its vicinity. This means,each of the engineered heart tissue rings used for preparing themultiring heart muscle construct is tangent to at least a furtherengineered heart tissue ring. Preferably, the distinct EHT rings areheld via suspension points of a holding device. According to a verysimple embodiment of the invention, several force-generating engineeredheart tissue rings as defined herein are stacked, i.e. laid upon eachother to form a stack with a plurality of loops extending in differentdirections (see for example FIG. 8A). It is particularly preferred thatthe force-generating EHT rings are placed into contact by stacking ofthe distinct rings to form a central region in which theforce-generating engineered heart tissue rings overlap with each otheras shown in FIG. 8A). According to an alternative embodiment severalrings are placed into contact as shown in FIG. 8B. Apart from that, anyother random distribution of the tissue rings in which each ring is incontact with at least another one are possible. It has been found incourse of the invention, that the tissue rings which have beenpositioned into contact in this manner fuse and form a complex in-unisoncontracting construct which is synchronized with respect to itscontracting activity. This finding allows for the generation of largetissue-like structures by fusing distinct contractile EHT rings (orprefused multiring structures comprising several of theses EHT rings).

In a further step, the force-generating engineered heart tissue rings,which are in contact with each other, are cultured under conditionswhich allow the fusion of the contacted ring structures to form anartificial heart muscle construct. For this purpose, the engineeredheart tissue rings are normally incubated in culture dishes, wherein therings are submersed in appropriate culture medium. If a holding deviceis used for culturing, the device should be designed to allow the tissuerings to stay in continuous contact with the culture medium. The choiceof the culture medium is not critical. Conventional media for culturingcardiac myocytes may be used. Such media are known in the art anddescribed, for example, in Zimmermann, W. H. et al., Circulation 106, I151-7 (2002) and published International Application WO 01/55297. Thesemedia may further be modified, i.e. by substituting Matrigel against amixture of Insulin (10 pg/ml) and triiodothyronine (1 nM). It was foundthat the addition of insulin and triiodothyronine allowed for thegeneration of strongly contracting EHTs. Simultaneous addition of bothfactors for only 24 h at the beginning of the EHT culture wassufficient. By using insulin and triiodothyronine during EHTconstruction, improved EHT contractility was achieved. These effects didnot stem from enhanced overall EHT cell number, since no difference inthe DNA content and no apparent structural differences were observed.Rather, the effects may be the result of enhanced protein content orimproved survival of cardiomyocytes leading to a higher cardiomyocytefraction without an apparent change of the overall cell number (standardEHTs on culture day 12 contain roughly 100.000 cardiomyocytes and atotal cell number of 600.000; a fractional increase in cardiomyocytenumber may have significant effects on force of contraction, may lead toan increase in overall “cardiomyocyte” protein [e.g. sarcomeric actin]but may not lead to a measurable [by total DNA quantification and cellcounting after enzymatic digestion of EHTs) increase in total cellnumber in EHTs treated with insulin and triiodothyronine. Furthermore,the media as used herein for culturing stem cells in example 6 can beused as well.

According to a more preferred embodiment of the invention, the samemedia which has been used for casting the EHT rings are used duringincubation of the force-generating engineered heart tissue rings forfusion (see examples 1 and 6 for neonatal cardiomyocyte and stem cells,respectively). Suitable culturing conditions are described in the artand comprise a physiological temperature range of 30−40° C., preferably3638° C., and more preferably 37° C. The percentage of 0₂ in the ambientair should range from 21-80%, preferably 70%, 60% 50%, and morepreferably 40%. Culturing may be performed for 3 to 50 days, preferably10-30 days, and more preferably 15-20 days.

According to preferred embodiment of the present invention a pluralityof distinct force-generating engineered heart tissue rings are placedinto contact to form the fused multiring EHT construct. Preferably, fiveor more tissue are placed into contact, for example six, seven, eight,nine, ten, eleven, twelve or more. More preferably, more than 15, 20,30, 40, 50, or even up to 100 force-generating engineered heart tissuerings are placed into contact to form the fused multiring EHT construct.It has to be understood that larger multiring EHT constructs can also beformed in several steps, for example by forming several constructs of5-10 EHT rings in a first step and fusing these different constructs ina subsequent step. In any case, care should be taken that the resultingmultiring EHT construct is cultured for a further period of time toallow synchronization of contractile activities.

According to preferred embodiment of the present invention, theforce-generating engineered heart tissue rings used for preparing themultiring heart muscle construct of the invention are subjected totensile stress prior to or simultaneously with placing into contact theat least two force-generating engineered heart tissue rings. Forexample, when using a device of the invention as described in moredetail below, placing into contact of the EHT rings and subjecting totensile stress can be performed in one step. Subjecting to tensilestress can for example be performed by actively stretching the tissuerings, for instance by use of a mechanical stretching device like theone described in Zimmermann, W. H. et al., Circulation Res 90, 223-30(2002). Such device can provide for a “static” load, i.e. a permanentload produced by expanding the ring by 3 to 20%, preferably 10% of itsoriginal length (original length determined in the absence of any load)as described in WO 01/55297. Alternatively, a “phasic” load can beapplied by periodically expanding the ring by 3 to 20%, preferably 10%of its original length, for example at a frequency of 0.1-10 Hz,preferably 1-5 Hz. Such stretching can be regarded as a static or phasicmanner, respectively.

In several publications, it was demonstrated that contractile propertiesof artificially synthesized heart tissue rings can be improved bymechanically stretching the rings after there casting (see Eschenhagenet al., 2002; Fink et al., 2000; Zimmermann et al., 2002b). It has beendemonstrated that mechanical load improves both orientation anddifferentiation of muscle cells in EHT rings. Tensile stress can beapplied for a period of time ranging from several hours to more than 40days. Preferably, tensile stress is applied for 5-9, more preferably 7days. During imposing tensile stress, the EHT rings are cultured underthe conditions recited above.

According to a preferred embodiment of the invention, tensile stress isapplied by a static, phasic or auxotonic manner or a combinationthereof. According to a particularly preferred embodiment of theinvention, tensile stress is applied by a auxotonic manner. An“auxotonic manner”, as used herein, means that the engineered hearttissue rings are kept under conditions so that they have to contractagainst a defined force and are reexpanded during relaxation of the EHTring by another force or, preferably, the same force. For example, ifthe tissue ring is held under tension by using a spring, this springwill provide a defined force against which the EHT must contract. Uponrelaxation of the EHT ring, the spring will expand the EHT ring again.It has been found that culturing the EHT rings in this manner leads to aconsiderable increase of contractile properties compared to phasic andstatic processes (see FIG. 1B). Therefore, both the single engineeredheart tissue rings as well as the artificial multiring heart muscleconstruct consisting of the distinct heart tissue rings show improvedmaximal active forces during contraction. Under auxotonic load, the EHTrings have to perform contraction under conditions which resemble thesystole and diastole phase of the heart cycle. In other words, theengineered heart tissue rings are held under conditions whichcorresponds to the native environment of active heart tissue.

Auxotonic tensile stress can be applied, for example, by elasticallysuspending each of the at least two force-generating engineered hearttissue rings between at least two associated suspension means wherein atleast one of said associated suspension means is resiliently biased(i.e. mounted to be movable against a bias force) so that the individualforce-generating EHT rings are able to contract against the bias forceprovided by the suspension means and are reexpanded during relaxation ofthe EHT ring. Preferably each of the force-generating engineered hearttissue rings is suspended between two associated suspension means.Preferably, the two suspension means can be variably adjusted to be usedwith rings of different diameters. The suspension means may be part of adevice as explained in detail below. Such device comprises a pair ofsuspension means for each EHT ring. According to a further embodiment,all of the associated suspension means are resiliently biased.

Preferably, the at least one suspension means can be adjusted to varythe tensile stress. This means, the load applied to the EHT rings can beeasily adjusted to a specific strength by modulating the tensionprovided by the resilient means. For example, springs can be used asresilient means comprising spring coils which can be adjusted in orderto modulate the resilient tension of the spring.

As already mentioned, “auxotonic” tensile stress can be appliedsimultaneously with placing into contact of the at least twoforce-generating engineered heart tissue rings. For example, thedifferent EHT rings can be suspended in a device as explained in detailbelow so that the tissue rings are held in a position that provides fora plurality of contact points between the different heart tissue rings.It has been found that the fusion of the different rings at theircontact points occurs also during the different rings are subjected tothe tensile stress. Surprisingly, subjecting the heart tissue rings totensile stress in terms of auxotonic (or phasic or static) loadobviously does not hamper formation of a fused multiring construct.Therefore, the culturing which allows fusion of the at least twoengineered heart tissue rings to form an artificial heart muscleconstruct can be performed while the heart tissue rings are held undertensile stress.

According to a further aspect, the invention refers to multiring EHTconstructs which comprise at least two force-generating engineered hearttissue rings fused with each other to form an interconnected tissueconstruct. The multiring engineered heart tissue construct is obtainableaccording to a method described above. As already explained, themultiring constructs can comprise more than 5 EHT rings, for example 15,20, 30, 40, 50, or even up to 100. Preferably, each of the EHT rings hasan outer diameter of 8-12 mm.

Preferably, the multiring EHT constructs of the invention are comprisedof fused engineered heart tissue rings which are stacked to form acentral region in which the force-generating engineered heart tissuerings overlap with each other. A construct, which results from stackingof five different tissue rings is depicted in FIG. 8A. Further examplesof multir-ing EHT construct structures are provided in FIG. 8B and FIG.8C. FIG. 8B shows a plurality of ring structures which are suspended bya flexible lateral suspension device wherein the distinct ringstructures are in contact with each other in the region of contact withthe suspension means. It has to be understood by the person ordinaryskilled in the art that these examples of ring assemblies are notlimiting. A large number of further geometric structures are conceivablefor example bag- or net-shaped structures or different forms ofstackings.

The multiring heart muscle constructs provided by the present inventionshow properties which makes them particularly useful for tissue graftsin heart tissue augmentation and/or replacement therapy. In animalmodels, it has been shown that multiring heart muscle constructsgenerated from neonatal myocard cells of rats were suitable to providefunctional tissue grafts which could be successfully transplanted toinfarcted rat hearts and support dysfunctional tissue damaged aftermyocard infarct (see examples). The multiring engineered heart tissueconstruct comprise cardiac myocytes, i.e. cells comprising one or moreof the following proteins: cardiac myosin heavy chain, α-actinin, desminand/orcardiac troponin I. It is clear that tissue grafts which areintended to be used in human therapy should be derived from cells ofhuman origin. Therefore, according to preferred embodiment of thepresent invention, the multiring heart muscle constructs are comprisedof force-generating engineered heart tissue rings derived from humancells. The human origin of the EHT can be confirmed by a plurality ofdifferent methods known in the art, for example by amplification ofspecies-specific DNA sequences and/or the antibody-based detection ofspecies-specific antigens, such as polypeptides. The preparation of theengineered heart tissue rings starting from human stem cells isexemplified in detail in example 6 of the present invention.

It is preferred that the multiring EHT construct of the invention has aoverall twitch tension of more than 2.5 mN, more preferably more than 3mN, for example 3.5 mN, 4 mN, 4.5 mN, 5 mN, 5.5 mN, 6 mN, 10 mN, 15 mNor more. The capability of the multiring construct to contract can bedetermined in accordance with methods as described in the prior art, forexample, by monitoring the constructs in standard organ baths (seeZimmermann et al., Biotech. Bioeng., 68, (2000)).

According to a further aspect, the invention relates to the use of themultiring EHT construct of the invention for drug target validation anddrug development. According to a preferred embodiment, the multiring EHTconstruct is used in drug screening or target validation assays. In thiscontext, it is preferred that the multiring EHT construct used isderived from a mammal, such as a human. For example, the multiringconstruct can be used to analyze the capability of candidate drugs tointerfere with the physiological function of the native cardiac tissueof a mammal. For this, the capability of said candidate drug to enhanceor reduce contractile functions of the multiring EHT construct isdetermined. Moreover, the multiring EHT construct may be used indetermining the influence of the activity of certain genes on thephysiological function of the native cardiac tissue of a mammal(referred to herein as target validation). For this purpose, genes ofthe cells within the distinct EHT rings (or cells which are used forpreparing the same) may be knocked-out or overexpressed or otherwiseinfluenced in view of their expression rate (e.g. by addition ofinhibitory molecules, such as small interfering RNAs). Genes may beselectively switched on or off, and the alterations in the contractilefunctions of the multiring EHT construct are determined.

The invention provides a method for providing force-generatingengineered heart tissue rings derived from human cells comprising thesteps of:

-   a) providing undifferentiated human embryonic stem cells;-   b) culturing said undifferentiated human embryonic stem cells under    conditions which allow for propagating the cells;-   c) mixing the cells with a suitable scaffold material in a circular    casting mold;-   d) culturing the human embryonic stem cells under conditions which    allow for differentiation of the cells into cardiac myocytes and    formation of a tissue ring structure.

The method can be performed by using the cell lines, media andconditions described in example 6 of the present invention.Particularly, the embryonic stem cells may comprise a selection systemand may be subjected to selection before or simultaneously withreconstitution of the EHTs. As explained in more detail below, asuitable scaffold material may be collagen type I. As a matter ofcourse, the human force-generating engineered heart tissue ringsresulting from the method may also be subjected to tensile stress asdescribed above with respect to the multiring constructs.

Accordingly, the invention further provides a force-generatingengineered heart tissue ring derived from human cells, preferably humanstem cells. The tissue ring essentially consists of differentiated humancardiac myocytes and other cell types usually found in heart tissue,such as fibroblasts, smooth muscle cells, endothelial cells, cells ofleukocytic origin, like macrophages, etc.) which are embedded by anon-cellular matrix. The presence of cardiac myocytes may be confirmedby detecting one or more of the following proteins: cardiac myosin heavychain, α-actinin, desmin and/orcardiac troponin I.

According to a preferred embodiment, the force-generating engineeredheart tissue ring derived from human cells is used in drug screeningassays. For example, the EHT ring derived from human cells can be usedto analyze the capability of candidate drugs to interfere with thephysiological function of the native cardiac tissue of a mammal. Forthis, the capability of said candidate drug to enhance or reducecontractile functions of the human EHT is determined. Moreover, theforce-generating engineered heart tissue ring derived from human cellsmay be used in determining the influence of the activity of certaingenes on the physiological function of the native cardiac tissue of amammal (referred to herein as target validation). For this purpose,genes of the cells within the EHT ring (or cells which are used forpreparing the same) may be knocked-out or overexpressed or otherwiseinfluenced in view of their expression rate (e.g. by addition ofinhibitory molecules, such as small interfering RNAs). Genes may beselectively switched on or off, and the alterations in the contractilefunctions of the human EHT are determined.

According to a further aspect, the invention provides a device forpreparing a multiring engineered heart tissue construct, comprising aplurality of associated suspension means for suspending force-generatingengineered heart tissue rings under tensile stress, wherein at least oneof each of the associated suspension means is resiliently biased so thatthe suspended force-generating engineered heart tissue ring is able tocontract against the bias force provided by the suspension means,wherein the suspension means are arranged to each other so that eachforce-generating engineered heart tissue ring has one or more contactpoints to an adjacent force-generating engineered heart tissue ring whensuspended in the device. A preferred embodiment of a device according tothe invention is depicted in FIG. 10.

Preferably, two suspension means are associated to each other to form apair of associated suspension means. It is preferred that the devicecomprises more than two pairs of associated suspension means so thatmore than two EHT rings can be suspended in the device. Preferably, thedevice is design for more than 5 EHT rings, for example 15, 20, 30 oreven more.

According to a further embodiment, the associated suspension means ofthe device are arranged to each other so that the force-generatingengineered heart tissue rings can be suspended by stacking to form acentral region in which the force-generating engineered heart tissuerings overlap with each other.

According to a particularly preferred embodiment, all associatedsuspension means are resiliently biased. Preferably, at least onesuspension means which is resiliently biased can be adjusted to vary thetensile stress.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention is described by way of examples withreference to the accompanying drawings in which:

FIGS. 1A-1F show the influence of the present amount of oxygen (FIG. 1A)of the applied load (FIG. 1B) and of the amount of insulin present (FIG.1C) as well as a single EHT ring (FIG. 1D) a multiring engineered hearttissue construct suspended in a suspension device (FIG. 1E) and theresulting multiring heart tissue construct (FIG. 1F);

FIGS. 2A-2F show the multiring EHT construct while being implanted (FIG.2A) and four weeks after engraftment at different magnification scales(FIG. 2B);

FIGS. 3A-3K show the identification of grafted cells by in vitro DAPIlabelling;

FIGS. 4A-4C show the epicardial activation time in the area ofinfraction (FIG. 4A) and in hearts with grafts (FIG. 4B), and the totalactivation time in right, anterior, lateral and posterior segments ofthe hearts (FIG. 4C);

FIGS. 5A-5F show the changes in left ventricular function aftermultiring EHT construct implantation for various parameters;

FIGS. 6A-6D show the influence of multiring EHT construct grafting onleft ventricular hemodynamics for various parameters;

FIGS. 7A-7C show the results from echocardiography (FIG. 7A), magneticresonance imaging (FIG. 7B) and hemodynamic analysis (FIG. 7C),

FIGS. 8A-8C show schematic representations of different geometries ofmultiring EHT constructs according to the invention;

FIG. 9A-9D show the generation of different EHT geometries prepared inthe course of the invention.

FIG. 10 shows a holding device which may be employed for suspending amultiring EHT construct while carrying out the invention.

FIG. 11 shows the composition of a serum-free culture medium suitablefor culturing cardiac myocytes and EHTs under chemically definedconditions.

FIGS. 1A-1F show the influences of oxygen, load, and insulin inisometric contraction experiments in single engineered heart tissue(EHT) rings (TT: twitch tension). Direct comparison of calcium responsecurves revealed that EHT ring benefited from oxygen supplementation (0₂:n=11 at 21%; n=5 at 30%; n=10 at 40%; FIG. 1A), auxotonic (aux.; n=8) incontrast to static (Sta.; n=8) or phasic (Pha.; n=8) load (FIG. 1B), andaddition of insulin (10 fag/ml; n=4) when compared to untreated controls(n=3; FIG. 1C). To increase size, 5 EHT rings (FIG. 1D) were stacked ina custom made device that facilitate EHT fusion and contractions underauxotonic load (FIG. 1E), resulting in synchronously contractingmultiring EHT constructs (FIG. 1F) ready for in vivo engraftment. *P<0.05 vs. 21% 0₂, static load, and culture in the absence of insulin(Ctr.), respectively by repeated ANOVA. Bars: 10 mm.

FIGS. 2A-2F show the morphology of grafted multiring EHT construct 4weeks after engraftment. Multiring EHT constructs were implanted througha left lateral thoracotomy (FIG. 2A). Mid-ventricular cross sections ofexcised hearts 28 days after implantation revealed that engraftedconstructs remained pale (FIG. 1B) but were firmly attached to theinfarct scar (c). H&E staining of paraffin sections through theinfarcted left ventricular free wall 4 weeks after construct engraftmentdemonstrated the formation of thick cardiac muscle on top of the infarctscar (FIG. 1D; multiring EHT construct encircled). High powermagnification of the infarct area showed that engrafted constructs formcompact and oriented heart muscle (FIGS. 1E-1F) spanning the transmuralinfarct.

FIGS. 3A-3K show the identification of grafted cells by in vitro DAPIlabelling. Bright light (FIG. 3A) and UV-illuminated (FIG. 3B) imagesdepict a DAPI labelled multiring EHT construct prior to implantation.Higher magnification revealed nuclear DAPI staining (FIG. 3C;magnification of marked area in FIG. 3B). Four weeks after engraftment,cells could readily be distinguished from native myocardium by the bluestaining of the donor cell nuclei (low power view; FIG. 3D: actin; FIG.3E: background; FIG. 3F; nuclei in EHT graft; FIG. 3G: merged image).High resolution laser scanning microscopy revealed the highlydifferentiated sarcomeric organization of engrafted cardiac myocytes(FIG. 3H: actin-green; nuclei-blue). Moreover, vascularization of graftswas evident (FIG. 3I, arrows in reconstituted confocal images; FIG. 3J,cross section; actin-green, nuclei-blue, actinin-red). Newly formedvessels contained many DAPI-positive cells (FIG. 3K, actin-green).Macrophages (ED2-red) with blue nuclei were found in close proximity tonewly formed vessels. Erythrocytes (*) were visualized by differentialinterference contrast imaging (FIG. 3K).

FIGS. 4A-4C depict results of the electrical integration analysis ofgrafted multiring EHT constructs in vivo. Epicardial activation time wasanalyzed in Langendorff-perfused hearts from experimental rats 4 weeksafter Sham surgery or multiring EHT construct implantation. 3D plots ofrepresentative activation times display the expected delay of epicardialactivation in Sham hearts in the area of infarction (FIG. 4A).Activation was restored to physiological values in hearts with grafts(FIG. 4B). Total activation time was assessed for statistical analysisin right, anterior, lateral, and posterior segments of the hearts (FIG.4C). * P<0.05 vs. Sham by ANOVA with Mann-U-Whitney test.

FIGS. 5A-5F show the changes in left ventricular function aftermultiring EHT construct implantation. Left ventricular end-diastolicdiameter (LVEDD; FIG. 5A) and fractional area shortening (FAS; FIG. 5B)determined by ECHO. “Healthy” indicates age-matched untreated controls(n=29). “Infarction” indicates pooled values (n=33) measured 14 daysafter infarction in those animals that underwent Sham (n=15) and EHT(n=18) surgeries (see FIG. 7A for individual group data). These animalswere reevaluated after additional 28 days by ECHO and subsequentlysubjected to MRI (FIGS. 5C-5F) with 2 and 3 exceptions in multiring EHTconstruct and Sham groups, respectively, due to technical reasons.Maximal and minimal volumes of the left ventricle were calculated from4D CINE mode MRI images in multiring EHT construct (n=15) and Sham(n=13) rats and supported the ECHO findings (FIG. 5D). To assess localcontractility, systolic thickening in anterior (AWThF; FIG. 5E) andposterior (PWThF; FIG. 5F) segments of the ventricular wall wereanalyzed. MRI data from healthy rats (n=29) and 2 weeks after infarction(n=8) were recorded in an independent series of experiments and aredisplayed for comparison. * P<0.05 multiring EHT construct vs. Sham byunpaired student's t-test. P<0.05 vs. “Infarction” by paired student'st-test.

FIGS. 6A-6D show the influence of multiring EHT construct grafting onleft ventricular hemodynamics. Multiring EHT construct engraftment (n=6)resulted in a leftward shift of the pressure-volume relationshipindicating an improvement of hemody-namics when compared to Sham (n=8)animals (FIG. 6A; representative pressure-volume loops). In detailanalysis of left ventricular enddiastolic volume (LVEDV) and pressure(LVEDP) as well as relaxation (tau) demonstrated a prevention of furtherdilation (FIG. 6B) and a normalization of LVEDP (FIG. 6C) and tau (FIG.6D) by multiring EHT construct implantation. CATH data from healthy rats(n=6) and 2 weeks after infarction (n=9) were recorded in an independentseries of experiments and are displayed for comparison. * P<0.05multiring EHT construct vs. Sham by unpaired student's t-test.

FIGS. 7A-7C show the results from echocardiography (FIG. 7A), magneticresonance imaging (FIG. 7B), and hemodynamic analysis (FIG. 7C).

FIGS. 8A-8C show different geometries of multiring EHT constructsaccording to the invention: (FIG. 8A) star-shaped multiring EHTconstruct prepared from 5 force-generating engineered cardiac tissuerings by stacking; (FIG. 8B) tubular multiring EHT construct prepared byplacing multiple EHT rings in series on a holding device to establishside-to-side contact followed by fusion and formation of synchronouslycontracting multiring engineered heart tissue construct; (FIG. 8C)alternatively, EHT rings may be cut to yield EHT stripes. The latter maybe stacked freely onto each other whilst being held at a defineddistance under defined load, which may be static, phasic or auxotonic.This format allows free organization of EHT rings into multiunitengineered heart tissue construct.

FIGS. 9A-9D demonstrate the generation of different EHT geometries. EHTsfuse after sustained contact to form in-unison contracting complexcardiac muscle constructs. Star-shaped EHTs (FIG. 9A) were generated bystacking 5 EHTs on a custom-made holder. Single-unit EHTs fused in thecenter. 5 EHTs (FIG. 9B) were grown on horizontal glass pipettes.Adjacent EHTs fused to form a tubular construct. 6 EHTs (FIG. 9C) werecut open and layered to form a contracting network. 3 EHTs (FIG. 9D)were twirled together to form a longitudinal “rope” structure. Bars: 10mm;

FIG. 10 shows a holding device which may be employed for the method forpreparing a multiring engineered heart tissue construct according to theinvention, in particular an embodiment of a suspension device forsuspending a star-shaped multiring EHT construct. There are twosemi-annular brackets. Each bracket comprises five suspension means, inthis example in the form of pins. Two opposing pins or suspension means,one on each of the two brackets serve to suspend one engineered hearttissue ring. The two brackets are connected to each other by twosprings. The two brackets may be moved closer with respect to each otheragainst the force of the two springs. The dimensions of the brackets andthe positions of the suspension means located thereon are chosen suchthat suspension of the engineered heart tissue rings is accompanied bymovement of the two opposing brackets closer to each other so that therings are suspended under a tensile load created by the springs locatedinbetween the brackets. In alternative arrangements each suspensionmeans could be mounted separately and a spring could be located betweena pair of opposing suspension means or pins in order to create a biasfor suspending a engineered heart tissue ring under tensile load aroundthe pair of suspension means. The engineered heart tissue rings aresubjected by the suspension device and can be adjusted by properly usingthe spring or springs acting between opposing suspension means.

FIG. 11 shows the composition of a serum-free culture medium which canbe used for culturing cardiac myocytes and EHTs under chemically definedconditions. The medium comprises several growth factors added assupplements. The skilled person will understand that one or more ofthese growth factors may be replaced against other growth factors oreven omitted from the medium without any adverse effect on the growth ofcardiac myocytes or the formation of EHTs.

EXAMPLES

Experimental animals were maintained in accordance with the guidingprinciples of the American Physiological Society. Data are presented asmean±standard error of the mean or box plots with mean and 95%confidence interval. Statistical differences were determined usingpaired and unpaired two-tailed Student's t-tests (in vivo data),repeated ANOVA (in vitro contraction experiment), or Mann-U-Whitney test(mapping). A P value of <0.05 was considered statistically significant.

Example 1 ETH Construction from Cardiac Myocytes of Neonatal Rats

EHTs were constructed as previously described in Zimmermann, W. H. etal., Circulation 106, I 151-7 (2002) and published InternationalApplication WO 01/55297. Briefly, EHT rings (reconstitution volume: 0.9ml) were prepared by mixing isolated heart cells from neonatal rats(2.5×10⁶ cells/EHT) with collagen type I from rat tails (0.8 mg/EHT; pHadjusted to physiologic values −7.4 with 0.1 N NaOH), serum-containingculture medium (2×DMEM, 20% horse serum, 4% chick embryo extract, 200U/mL penicillin, and 200 mg/mL streptomycin; similar volume asneutralized collagen), and Engelbreth-Holm-Swarm tumor exudate(“Matrigel” final concentration 10% v/v; tebu, France).

EHTs were transferred after 7 days of culturing in casting molds ontocustom made stretch devices to facilitate static (110% of slack length),phasic (from 100 to 110% of slack length at 2 Hz), or auxotonic (cultureon deflection coils at 110% of slack length adjusted to deflect 1 mm at1-1.5 mN) loading. Static loading denotes a condition of constant straininduced by its lateral attachment to stable holders. Phasic load isimposed on EHTs by attaching EHTs to mobile (e.g. motor driven) lateralholders that are forced to move apart to defined positions with adefined frequency. Auxotonic loading conditions are installed byattaching the EHTs to lateral deflection coils adjusted to be deflectedtowards a defined condition by a defined force. Taken together (1) thelateral distance can be permanently adjusted within a variable range ata defined time range to achieve static loading; (2) the lateral distanceof EHTs can be adjusted within a variable range at a defined time underphasic load; (3) the lateral distance of EHTs can be adjusted to adefined yet variable position depending on the intrinsic contractileactivity (force and frequency) by auxotonic loading. Contractileparameters were analyzed by isometric contraction experiments of singleloop EHTs as described in Zimmermann, W. H. et al., Circulation 106, I151-7 (2002) and published international application WO 01/55297.Multi-loop EHTs were generated by stacking single circular EHTs. 5 EHTs(each with an outer/inner diameter −10/8 mm; FIG. 1d ) were stacked in acrosswise manner on a specially designed holding device (FIG. 1e ) andincubated in the above medium (DMEM, 10% horse serum, 2% chick embryoextract, 100 U/mL penicillin, and 100 mg/mL streptomycin) under standardconditions (37° C., 5-101 CO₂, 21-40% 0₂) in a holding device accordingto the invention. Under these conditions circular EHTs fused and formedcontracting multiring EHT constructs (diameter −15 mm, thickness: 1-4mm; FIG. 1f ). Non-contractile grafts for control purposes wereconstructed according to the EHT protocol described in Zimmermann, W. H.et al., Circ Res 90, 223-30 (2002) Instead of freshly isolated heartcells rat cardiac non-myocytes (n=3) or vascular smooth muscle cells(n=2) were used.

In course of the invention, it was found that three modifications of theoriginal reconstitution protocol described in Zimmermann, W. H. et al.,Circ Res 90, 223-30 (2002); Fink, C. et al., Faseb J 14, 669-79 (2000)and in the published International Application WO 01/55297 enhancedcontracting characteristics of the resulting EHTs: (1) Increasingambience oxygen from 21% to 40% (FIG. 1a ), (2) culture under conditionsthat allow active contractions and relaxation (auxotonic strain; FIG. 1b), and (3) supplementation of the culture medium with 10 pg/ml insulin(FIG. 1c ). The use of insulin together with triiodothyronine (1 nM)enabled construction of EHT without Matrigel.

Example 2 Infarct Model and Grafting

The potential of multiring EHT constructs to repair diseased hearts inmale Wistar rats with myocardial infarcts was tested. Myocardialinfarctions were generated in ventilated, isoflurane (2%) anesthetizedmale Wistar rats (318+3 g; n=121) by permanent ligation of the leftanterior descending coronary artery (LAD ligation; 5-0, Prolene,Ethicon, Germany). 14 days after LAD ligation (first surgery) anindependent and blinded investigator evaluated infarct localization andsize by Echocardiography (ECHO).

Echocardiography was performed in volatile isoflurane (2%) anaesthesiaas described previously with a HP Sonos 7500 System (Philips, Amsterdam,The Netherlands) equipped with a 15 MHz linear array transducer(Zimmermann, W. H. et al., Circulation 106, I 151-7 (2002)). To monitorchanges in myocardial performance all animals were subjected to ECHO 14days post LAD ligation and again 4 weeks after grafting orSham-operation (longitudinal study design). Additionally, baselinefunction and dimensions in healthy age-matched Wistar rats (n=29) wereevaluated. ECHO recordings and analyses were performed by blindedinvestigators. Inter- and intraobserver variation of reported data was<10%.

Given the well-known variability of infarct size after LAD ligation inrodent models, a transmural infarction with a fractional area shortening(FAS) of less than 40% was defined as inclusion criterion (FAS of −60%was detected in 29 healthy Wistar rats of comparable age). Multiring EHTconstructs were implanted 2 weeks after infarct induction by suturingmultir-ing EHT constructs onto the epicardium (6 single knots; 5-0,Prolene, Ethicon, Germany). Fixation on healthy myocardium was ensuredwhile the center of the multiring EHT construct was arranged above theinfarct. In the Sham group, sutures were placed as if multiring EHTconstructs were implanted. All animals received immunosuppressants asdescribed previously in Zimmermann, W. H. et al., Circulation 106, I151-7 (2002), i.e. azathioprine: 2 mg/kg bodyweight/day, ciclosporin A:5 mg/kg bodyweight/day, methylprednisolone: 5 mg/kg bodyweight/day.

51 rats did not survive the LAD ligation, 19 rats did not display anyfunctional deficits, and 10 rats had a FAS >40% after LAD ligation.Eventually, 41 rats (FAS <40%) were subjected to engraftment (n=24; FIG.2a ) or Sham operation (n=17). 4 from 24 (EHT group) and 2 from 17 (Shamgroup) rats died during the second surgery. Two additional rats from theEHT group died during the 4 week study phase after the second surgery.Hence, 18 and 15 animals from EHT and Sham groups, respectively,survived the complete study duration. These animals were subjected toepicardial mapping, echocardiography, magnetic resonance imaging, LVcatheterization and morphological studies 4 weeks after the secondsurgery.

Example 3 Epicardial Mapping

Electrical coupling of multiring EHT constructs to the host myocardiumwas assessed 4 weeks after engraftment by high resolution epicardialmapping as described in Dhein, S. et al., Circulation 87, 617-30 (1993).Briefly, hearts were excised and Langendorff-perfused with Tyrode'ssolution at a constant pressure of 70 cm H₂O and 37° C. Unipolar ECGswere recorded simultaneously from 256 AgCl electrodes (interelectrodedistance: 1 mm; sampling rate: 4 kHz/electrode; HALO system; P. Rutten,Hamburg, Germany) arranged in 4 polyester blocks around thecircumference of spontaneously beating hearts. Activation time wasdetermined at each electrode and the spread of excitation was analyzedby constructing isochrones. For quantitative analysis the totalactivation time assessed as the delay between activation of the firstand activation of the last electrode for each region under investigationwas determined.

Sham animals (n=5) demonstrated the expected delay of impulsepropagation in infarcted areas (FIGS. 4A and 4C). In contrast,epicardial activation was normal in rats that received multiring EHTconstruct grafts (n=8), indicating undelayed anterograde coupling ofmultiring EHT constructs to the host myocardium (FIGS. 4B-4C). Inaddition, the epicardial activation in animals that receivednon-contractile grafts containing either rat fibroblasts (n=2) or smoothmuscle cells (n=2) was studied. Here, similar conduction deficits as inthe Sham group were observed, i.e. an activation delay of 10 to 18 ms inthe lateral wall and no active responses in most areas of thefibro-blast/smooth muscle cell containing grafts.

Example 4 Functional Consequences of Grafting

Prior to our grafting studies, myocardial dimension and function wereassessed in an independent healthy age-matched control group by ECHO(n.29), MRI (n=29) and CATH (n=6). ECHO was performed as outlined above.Eventually, ECHO was performed 14 days after LAD ligation in all ratsthat survived the first surgery (n=70). 33 animals survived the completestudy and were subjected to ECHO (in 33 from 33 animals), MRI (in 28from 33 animals), and CATH (in 14 from 33 animals) 28 days after thesecond surgery. The results are depicted in FIGS. 7A-7C

Magnetic resonance imaging was performed in volatile isoflurane (2%)anaesthesia with a Bruker 4.7T Biospect System using a fast gradientecho sequence with TR 21 ms, TE 5 ms and a flip angle of 30 degrees.Recordings were ECG− and breath triggered. A total of 6-8 subsequentmovie frames were acquired with 256×128 pixels at 200×400 μm pixelresolution. A longitudinal view was obtained for orientation purposes.Based on the latter 20-30 cross sections (short axis) from apex to basewere imaged (4D cine movie). Subsequently, datasets were subjected tooff-line analysis with manual segmentation of the heart contours.Examiners were blinded to the study protocol.

For LV-catheterization, pressure-volume loops were recorded underisoflurane (2%) anaesthesia with a Millar 2 Fr catheter (model: SPR-838)connected to Aria/PowerLab data acquisition hardware (Millar/PowerLab)by a blinded investigator. Volume calibration was performed by equatingcatheter-recorded minimal and maximal conductance with minimal andmaximal MRI-volumes, respectively. All data were analyzed off line withPVAN 3.2 software (Millar) by a second blinded investigator.

In contrast to ECHO (longitudinal study design), it was refrained fromrepeated MRI and CATH due to high mortality of animals that underwentmultiple 4D CINE mode MRI (1-2 hours imaging time under anaesthesia) andthe availability of only one defined access to the left ventriclethrough the right carotid artery for CATH under closed chest conditions.Evaluation 14 days after infarction revealed marked enlargement of leftventricle dimension and volume (FIGS. 5A and 5D; FIG. 6B), a reducedfractional area shortening (FAS; FIG. 5B), a decreased anterior wallthickening fraction (AWThF; FIG. 5E) without notable effects on theposterior wall thickening fraction (PWThF; FIG. 5F), and a rightwardshift of the pressure-volume relationship (FIG. 6A). At the same time,left ventricular enddiastolic pressure (LVEDP; FIG. 6C) and tau (indexof relaxation; FIG. 6d ) remained almost normal indicating that theexperimental animals were in a compensated state of heart failure priorto the second surgery (FIG. 7A-7C). 28 days later, ECHO, MRI, and CATHdata collectively indicated further deterioration in Sham operatedanimals (FIGS. 5A-5F and 6A-6D; FIG. 7A-7C). In particular, leftventricular end diastolic dimensions (LVEDD) increased (FIG. 5A) and FASslightly decreased (GIF. 5B). Moreover, LVEDP and tau increased markedlyto >20 mmHg and 20 ms, respectively (FIGS. 6C-6D). In contrast, leftventricle dimensions and FAS remained unchanged in animals from the EHTgroup, i.e. they did not differ from baseline values 2 weeks after LADligation (FIG. 5A-5B). Whereas ECHO examination did not allow forunambiguous identification of volumes and active systolic thickening ofthe ventricular wall, the higher spatial resolution of MRI enabled us toprecisely visualize left ventricular volumes (from multiplane images andoffline 3D rendering of the total ventricle) and systolic wallthickening in anterior (area of infarct) and posterior (no infarct)segments of the left ventricle (FIGS. 5C-5F). MRI supported the ECHOdata and showed significantly smaller left ventricular volumes in theEHT group when compared to Sham animals (FIG. 5D). In addition, MRIdemonstrated a significantly improved AWThF (FIG. 5E), whereas PWThF wasnot affected (FIG. 5F). To assess whether non-myocytes or the plainphysical influence of multiring EHT construct grafting might havecontributed to the improvement of AWThF, the effect of non-contractilefibroblast (n=3) and smooth muscle cell grafts (n=2) was studied. As inthe Sham group and in contrast to the EHT group AWThF was markedlyreduced (−3±7%) and PWThF was not affected (31+131) in animals thatreceived non-contractile grafts (pooled data; n=5). CATH allowed fordetailed analysis of hemodynamic function with superior temporalresolution (FIGS. 6A-6D). Pressure-volume loop analysis supported theformer data showing that left ventricular dilatation was less aftermultiring EHT construct engraftment than after Sham surgery (FIG. 6B).Most strikingly, LVEDP and tau in rats with grafts did not differ fromhealthy controls (FIGS. 6C-6 D).

Example 5 Multiring EHT Construct In Vivo Morphology after Grafting

Four weeks after grafting, multiring EHT constructs could readily beidentified by its distinct appearance and location (FIGS. 2B-2C). Thepale color indicated that the multiring EHT construct retained a highconnective tissue content. Hearts were fixed in neutral buffered 4Wformaldehyde/1% methanol, pH 7.4 and subjected to histological analysisas described previously in Zimmermann, W. H. et al., Circulation 106, I151-7 (2002). Briefly, paraffin embedded sections (4 μm) were stainedwith hematoxylin and eosin (H&E; see FIGS. 2A-2F). Cryo-sections (10 μm)were stained with phalloidin-Alexa 488 to label f-actin and antibodiesdirected against a-sarcomeric actinin (1:1000; Sigma) and ED2 (fullstrength; Serotec) with appropriate secondary antibodies (see FIGS.3A-3K).

H&E staining of paraffin sections revealed formation of compact and welldifferentiated multiring EHT construct-derived heart muscle covering theinfarcted myocardium (FIGS. 2D-2F). Notably, the reconstitutedmyocardium was oriented along the circumference of the heart andconsisted of multiple cell layers with a total diameter of up to 1000 μm(FIG. 2E), a thickness never observed during in vitro culture.

To further evaluate whether cardiac myocytes within the reconstitutedmyocardium were of donor origin, multiring EHT constructs (n=5) werelabelled with DAPI (1 dig/ml) prior to implantation (FIGS. 3A-3C) andsearched for DAPI-positive cells after additional 4 weeks. Engraftedmultiring EHT constructs could be clearly distinguished from therecipient's myocardium by the blue fluorescence of the DAPI-labellednuclei (FIG. 3D-3G). In high power magnification, it was observed thatmultiring EHT construct-derived cardiac myocytes developed adifferentiated and well organized phenotype including in registryorganization of sarcomeres (FIG. 3H). DAPI-positive cardiac myocyteswere not observed in the host myocardium. DAPI-negative cardiac myocyteswere not noticed within the graft. Besides cardiac myocytes, manynon-myocytes showed blue nuclei (FIGS. 3I-3K). Interestingly, bloodvessels in the grafts contained cells with blue nuclei, suggestingdonor-origin (FIGS. 3I-3K). The presence of erythrocytes provedconnection of these vessels to the recipient's vasculature (FIG. 3K).

Example 6 ETH Construction from Human Embryonic Stem Cells 1. SuitableES Cell Lines

Pluripotent human embryonic cell lines have been established and can becommercially obtained from different providers. These cell lines havebeen listed by the US National Institutes of Health (Bethesda, Md.,USA), from which further information as to the cell line characteristicsand providers may be obtained. The following ES cell lines are availableand may be used for preparing EHTs: cell lines hESBGN-01, hESBGN-02,hESBGN-03 from BresGen, Inc. (Athens, USA), NIH Code: B001, BG02, BG03;cell lines Sahlgrenska 1, Sahlgrenska 2 from Cellartis AB (Goteborg,Sweden), NIH Code: SA01, SA02; cell lines HES-1, HES-2, HES-3, HES-4,HES-5, HES-6 from ES Cell International (Singapore), NIH Code: ES01,ES02, ES03, ESO4, ES05, ES06; cell line Miz-hES1 from the MedicalResearch Center, MizMedi Hospital (Seoul, Korea), NIH Code: MI01; celllines I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3, J 3.2 from the RambamMedical Center (Haifa, Israel), NIH Code: TE03, TE32, TE33, TE04, TE06,TE62, TE07, TE72; cell lines HSF-1, HSF-6 from the University ofCalifornia, Department of Obstetrics, Gynecology & Reproductive Sciences(San Francisco, USA), NIH Code: UC01, UC06; cell lines H1, H7, H9, H13,H14 from the Wisconsin Alumni Research Foundation (Madison, USA), NIHCode: WA01, WA07, WA09, WA13, WA14.

Different groups have shown that human embryonic stem cell lines WA01(H1), WA07 (H7), WA09 (H9) including subclones H9.1, H9.2 and ES02 candifferentiate either spontaneously or if co-cultured withvisceral-endoderm like cells (END-2) or liver parenchymal carcinomacells (HepG2) into cardiac myocytes (Xu, C., et al. Characterization andenrichment of cardiac myocytes derived from human embryonic stem cells.Circ Res 91, 501-8 (2002); Kehat, I. et al. supra; Mummery, C. et al.Differentiation of human embryonic stem cells to cardiac myocytes: roleof co-culture with visceral endoderm-like cells. Circulation 107,2733-40 (2003)). However, the propensity of mammalian embryonic stemcells to differentiate into cardiac myocytes is an inherent trait of alltrue embryonic stem cells and thus not limited to the above mentionedcell lines. Apart from embryonic cells, other stem cells may be used forpreparing the EHT rings or multiring constructs according to theinvention. Such stem cells include, but are not limited to, humanpluripotent adult stem cells with a propensity to give rise to cardiacmyocytes (e.g. c-kit positive cells (Beltrami et al. Cell 2003),endothelial progenitor cells (Badorff et al. Circ Res 2002), sidepopulation cells (Jackson et al. JCI 200x), Sca-1 positive cells (Oh etal. 2002), Is1-1 positive cells (Laugwitz et al. 2005). These cells maybe derived from the blood, bone marrow or from various organs includingthe heart, the liver, umbilical cord, placenta, fat tissue, amongothers.

It is particularly preferred to employ genetically modified humanembryonic stem cells for reconstitution of EHTs, e.g. cells capable ofexpressing proteins that allow selection of cardiac myocytes and/ordepletion of non-myocytes. By selecting ES, homogenous cell populationscan be formed which may again be substituted with defined and alsoselectable non-myocytes which include but are not limited to endothelialcells, fibroblasts, smooth muscle cells, and macrophages within the EHTrings. Moreover, selection allows for depletion of tumorigenic cellswhich is a prerequisite for the use of the EHTs in therapeuticapproaches. Selection can be performed before or simultaneously withculturing the cells in casting molds. For instance, invitro-differentiation may be performed directly after propagation of theES cells and the resulting embryonic stem cell derived cardiac myocytescan be subjected to selection before reconstituting the EHTs.Alternatively, undifferentiated embryonic stem cells are propagated andused for the preparation of EHT rings or multiring EHT constructs andselection is performed on the EHT constructs at a later stage.Preferably, selection is to be performed after the EHT rings or themultiring EHT constructs have been formed.

Selection may be achieved by stable introduction of selectable markergenes. The expression of such genes is controlled either by a cardiacmyocyte specific promoter (positive selection) or a non-myocyte specificpromoter (negative selection) into embryonic stem cells. These may bebut are not limited to:

-   -   amino-3′ glycosyl phosphotransferase conferring a neomycin        resistance [neoR]    -   herpes simplex virus thymidine kinase (TK] which transforms        ganciclovir into a cytotoxic triphosphate purine nucleoside        phosphorylase [PNP] from Escherichia coli which converts        fludarabine to toxic fluoro-ATP fluorescing proteins and        P-galactosidase which facilitate optical identification

Expression of neoR, fluorescing proteins, or P-galactosidase under thecontrol of a cardiac myocyte specific promoter, e.g. α-myosin heavychain promoter (see Klug, M. G., Soonpaa, M. H., Koh, G. Y. & Field, L.J. Genetically selected cardiac myocytes from differentiating embryonicstem cells form stable intracardiac grafts. J Clin Invest 98, 216-24(1996)), myosin light chain 2v promoter (see Muller, M. et al. Selectionof ventricular-like cardiac myocytes from ES cells in vitro. Faseb J 14,2540-8 (2000)) enable positive selection of cardiac myocytes byeliminating non-myocytes either through addition of cytotoxic G418 orfluorescence activated cell sorting (FAGS). The techniques for selectionare well known to the person of skill. Alternatively, non-myocytesexpressing TK or PNP under the control of a promoter that is not activein cardiac myocytes, e.g. the Oct-4 promoter (Boiani, M., Kehler, J. &Scholer, H. R. Activity of the germline-specific Oct4-GFP transgene innormal and clone mouse embryos. Methods Mol Biol 254, 1-34 (2004)), theRex-1 promoter (see Hosler, B. A., Rogers, M. B., Kozak, C. A. & Gudas,L. J. An octamer motif contributes to the expression of the retinoicacid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinomacells. Mol Cell Biol 13, 2919-28 (1993)) can be eliminated (negativeselection) by addition of ganciclovir or fludarabine, respectively(Lockett, L. J., Molloy, P. L., Russell, P. J. & Both, G. W. Relativeefficiency of tumor cell killing in vitro by two enzyme-prodrug systemsdelivered by identical adenovirus vectors. Clin Cancer Res 3, 2075-80(1997)). Genetic modification of human embryonic stem cells is eitherperformed by stable transformation utilizing established lipofection orelectroporation protocols to introduce the genetic information (cDNA]into stem cells (see, for example, Nagy, A., Gertsenstein, M.,Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: ALaboratory Manual. (2002); Eiges, R. et al. Establishment of humanembryonic stem cell-transfected clones carrying a marker forundifferentiated cells. Curr Biol 11, 514-8 (2001)) or by lentivirusmediated gene transfer leading to stable transduction of embryonic stemcells (see Ma, Y., Ramezani, A., Lewis, R., Hawley, R. G. & Thomson, J.A. High-level sustained transgene expression in human embryonic stemcells using lentiviral vectors. Stem Cells 21, 111-7 (2003)).Integration of the exogenous genetic material is confirmed by standardmethods (e.g. southern blotting, polymerase-chain-reaction; seeSambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual.(2001)).

2. Preparing EHTs from Selectable, Undifferentiated ES Cells

Selectable human embryonic stem cells are used for reconstitution in anundifferentiated state. The undifferentiated human embryonic stem cellsare cultured for propagation in Dulbecco's modified Eagle's medium(DMEM) with 20% batch tested fetal calf serum or 20% serum replacement,1 mM L-glutamine, 0.1 mM P-mercaptoethanol, and 1% nonessential aminoacids on mitotically inactivated fibroblast feeders (see Xu, C., et al.supra; Kehat, I. et al., supra) until a sufficient cell number of about0.1-10×10⁶ cells is reached. All substances are provided by severalcompanies including Invitrogen. Human basic-fibroblast growth factor(bFGF, 4 ng/ml) is not essential but may be added to improve maintenanceof an undifferentiated state. Antibiotics (100 U/ml penicillin and 100pg/ml streptomycin) may also be added. Subsequently, undifferentiatedselectable embryonic stem cells (0.1-10×10⁶ cells) are mixed withscaffold material (e.g. collagen type I, concentration: 0.51.5 mg/ml)and growth promoting culture medium (see above). The totalreconstitution volume consists normally of 0.5-1 ml but may be reducedor increased depending on the respective demand. The reconstitutionmixture is filled into casting molds (e.g. the circular molds describedin the WO 01/55297) immediately after mixing. Under normal cultureconditions (e.g. 37° C., 5-10% CO₂, 21-40% 0 ₂) solid tissue-likestructures will form within 1-10 days. Under these conditions,differentiation of the embryonic stem cells occurs spontaneously and mayadditionally be affected by exogenous factors (e.g. addition to theculture medium of growth factors and cytokines including insulin,insulin-like growth factor, cardiotrophin-1, bone morphogenic proteins,sonic hedgehog at EC₅₀ concentrations). The solid tissue-like structuresmay optionally be transferred on stretch devices (as described above forthe preparation of multiring constructs, or as described in Zimmermannfor EHT rings) to optimize culture medium perfusion and impose definedmechanical strain which may be static, phasic, or auxotonic—on thetissue constructs.

Stacking of human multiring engineered heart muscle may be performed asdescribed for the rat model during the differentiation and selectionprocess. EHT rings or multiring EHTs are composed of interconnectedmuscle strands consisting of interconnected single cardiac myocytes.Cell connections consist of gap junctions staining positive for connexinproteins. Single cardiac myocytes develop organized sarcomeres stainingpositive for a-sarcomeric actinin and other sarcomeric proteins. Themuscle function of EHT rings or multiring EHT constructs can be analyzedby isometric contraction experiments to demonstrate its organotypicresponsiveness for example to increasing calcium concentrations(positive inotropic effect) and isoprenaline (positive inotropic andlusitropic effects).

Positive or negative selection (see above) may be performed afterformation of contractile areas within the EHT rings i.e. 5-30 days aftercasting of the reconstitution mixture into the molds. Alternatively,selection may be performed after the rings have been transferred to thestretching device or during stacking for preparing the multiring EHTconstruct. Selection may be performed until optimal tissue structure andfunction has developed (e.g. 10-40 days depending on the observedeffect). Selection may be achieved by addition of a definedpharmacological agent (e.g. G418, ganciclovir, fludarabine) into theculture medium).

3. Preparing EHTs from ES-Derived Cardiac Myocytes

For the preparation of EHTs from human cells, we utilized cardiacmyocytes derived from an established human embryonic stem cell line (H9,subclone H9.2). However, it is to be understood that all true human andnon-human embryonic stem cells can be used for preparing the EHTs of theinvention. For example, human embryonic stem cells derived fromindividual blastomeres (Klimanskaya I, Chung Y, Becker S, Lu S J, 'LanzaR (2006) “Human embryonic stem cell lines derived from singleblastomeres”, Nature, electronic publication ahead of print on Aug. 23,2006) or from the inner cell mass of an embryo in the blastocyst stage(Thomson J A et al. (1998), Science 282:1145-7) as well as othermultipotent embryonic-stem cell like cells, e.g. from parthenogeneticembryos (Vrana K E et al. (2003), Proc Natl Acad Sci USA, 100 Suppl1:11911-6), adult germline stem cells (Guan K et al. (2006), Nature440:1199203), somatic nuclear transfer derived embryos (Wakayama T etal. (2001), Science 292:740-3) or reprogrammed somatic cells (Cowan C A,et al. (2005), Science 309:1369-7; Takahashi K and Yamanaka S (2006),Cell 126:663-76) can be used. Similarly, somatic stem cells (Badorff C,et al. (2003), Circulation 107:1024-32; Beltrami A P, et al. (2003),Cell 114:763-76; Dimmeler S, et al. (2005); J Clin Invest 115:572-83;Murry C E, et al. (2005), Circulation 112:3174-83) with a cardiogenicpotential are applicable in EHT construction as outlined for embryonicstem cells. Embryonic stem cell derived cardiac myocytes were selectedby micro-dissection after spontaneous differentiation in vitro.Differentiation was performed in suspension culture, and the cells weresubsequently plated on gelatine coated culture dishes as previouslydescribed by Kehat, I. et al., J Clin Invest 108, 407-14 (2001). Beatingclusters containing embryonic stem cell derived cardiac myocytes wereexcised with a sharp, sterile glass or plastic pipette. Prior toreconstitution, ES-cell derived cardiac myocytes were enzymaticallydispersed in collagenase IV (Life Technologies Inc., 1 mg/ml for 20 min)or with trypsin/EDTA. ES-cell derived cardiac myocytes (10.000-15×10⁶dispersed cells/500 pl reconstitution mix, preferably 20.000-250.000dispersed cells/500 pl reconstitution mix) were reconstituted withcollagen, Ma-trigel and culture medium according to the establishedprotocol by Zimmermann W. H. et al., Circ Res 90, 223-30 (2002) and WO01/55297, with the exemption that the following culture medium was used:DMEM (preferably containing 80% knock-out DMEM [no-pyruvate,high-glucose formulation; Life Technologies, Inc.]), 20% fetal calfserum, 1 mM glutamine, 0.1 mM p-mercaptoethanol, 1W non-essential aminoacids. Matrigel can be replaced by a mixture of triidothyronine (1 nM)and insulin (10 pg/ml). The EHT reconstitution mixture was pipetted intocircular casting molds as described and the mixture was incubated toallow hardening. First contractions of single cardiac myocytes wereobserved after 24 hours of human EHT (hEHT) culture, larger cellaccumulations within hEHTs started to contract synchronously afterapprox. 1-3 days whereas in unison contractions of whole EHTs with 1-2Hz were observed after approx. 2-10 culture days.

1-24. (canceled)
 25. A device for preparing a multiring engineered hearttissue construct, comprising a plurality of associated suspension meansfor suspending force-generating engineered heart tissue rings undertensile stress, wherein at least one of each of the associatedsuspension means is resiliently biased so that the suspendedforce-generating engineered heart tissue ring is able to contractagainst the bias force provided by the suspension means, wherein thesuspension means are arranged to each other so that eachforce-generating engineered heart tissue ring has one or more contactpoints to an adjacent force-generating engineered heart tissue ring whensuspended in the device.
 26. The device of claim 25, wherein twosuspension means are associated to each other.
 27. The device of claim25, wherein all associated suspension means are resiliently biased. 28.The device of claim 25, wherein the at least one suspension means can beadjusted to vary the tensile stress.
 29. The device of claim 25, whereinthe associated suspension means are arranged to each other so that theforce-generating engineered heart tissue rings can be suspended bystacking to form a central region in which the force-generatingengineered heart tissue rings overlap with each other. 30-31. (canceled)32. The device of claim 26, wherein all associated suspension means areresiliently biased.
 33. The device of claim 26, wherein at least one ofthe two suspension means can be adjusted to vary the tensile stress. 34.The device of claim 26, wherein the associated suspension means arearranged to each other so that the force-generating engineered hearttissue rings can be suspended by stacking to form a central region inwhich the force-generating engineered heart tissue rings overlap witheach other.
 35. The device of claim 27, wherein the associatedsuspension means are arranged to each other so that the force-generatingengineered heart tissue rings can be suspended by stacking to form acentral region in which the force-generating engineered heart tissuerings overlap with each other.